In vitro characterization of radiolabeled monoclonal antibodies specific for the extracellular domain of prostate-specific membrane antigen

Prostate specific membrane antigen (PSMA) is a well-characterized cell surf ace antigen expressed by virtually all prostate cancers (PCas). PSMA has be en successfully targeted in vivo with In-111-labeled 7E11 monoclonal antibo dy (mAb; ProstaScint; Cytogen, Princeton, NJ), which binds to an intracellu lar epitope of PSMA. This work reports the irt vitro characterization of th ree recently developed mAbs that bind the extracellular domain of PSMA (PSM A(ext)). Murine mAbs J415, J533, J591, and 7E11 were radiolabeled with I-13 1 and evaluated in competitive and saturation binding studies with substrat es derived from LNCaP cells. J415 and J591 were conjugated to 1,4,7,10-tetr aazacyclododecane-N,N',N':N"'-tetraacetic acid labeled with In-111. The upt ake and cellular professing of these antibodies were evaluated in viable LN CaP cells. All four mAbs could be labeled with I-131 up to a specific activ ity of 350 MBq/mg with no or little apparent loss of immunoreactivity, Comp etition assays revealed that J415 and J591 compete for binding to PSMA(ext) antigen. J533 bound to a region close to the J591 binding epitope, but J53 3 did not interfere with J415 binding to PSMA. mAb 7E11 did not inhibit the binding of J415, J533, or J591 (or vice versa), consistent with earlier wo rk that these latter mAbs bind PSMA(ext) whereas 7E11 binds the intracellul ar domain of PSMA. Saturation binding studies demonstrated that J415 and J5 91 bound with a similar affinity (K(d)s 1.76 and 1.83 nw), whereas J533 had a lower affinity (K-d, 18 nM). In parallel studies, all four mAbs bound to a similar number of PSMA sites expressed by permeabilized cells (1,000,000 -1,300,000 sites/cell). In parallel studies performed with viable LNCaP cel ls, J415, J533, and J591 bound to a similar number of PSMA sites (i.e., 600 ,000-800,000 sites/cell), whereas 7E11 bound only to a subpopulation of the available PSMA sites (95,000 sites/cell). This apparent binding of 7E11 to viable cells can be accounted for by a 5-7% subpopulation of permeabilized cells produced when the cells were trypsinized and suspended. Up to five D OTA chelates could be bound to either J415 or J591 without compromising imm unoreactivity. A comparison of the cellular uptake and metabolic processing of the I-131- and In-111-labeled antibodies showed a rapid elimination of I-131 from the cell and a high retention of In-111. All four mAbs recognize d and bound to similar numbers of PSMAs expressed by ruptured LN-Cap cells (i.e., the exposed intracellular and extracellular domains of PSMA). By com parison to J415 and J591, J533 had a lower binding affinity. Both J415 and J591 recognized and bound to the same high number of PSMAs expressed by int act LNCaP. By contrast, 7E11 bound to fewer sites expressed by intact LNCaP cells (i.e., the exposed extracellular domain of PSMA). Both J415 and J591 are promising mAbs for the targeting of viable PSMA-expressing tissue with diagnostic and therapeutic metallic radionuclides.